Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with non-complementary DNA/RNA is disfavored, which prevents PNA ligation to occur on single base-mismatched templates. Different readout strategies of the ligation reaction such as HPLC, MALDI-TOF-MS and fluorecence monitoring are discussed, and examples for the detection of a point mutation within single stranded and PCR-amplified double stranded DNA are provided.
DNA template Native chemical ligation PNA, Single base specificity Templated chemistry
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Dose C, Seitz O (2008) Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes. Bioorg Med Chem 16:65–77PubMedCrossRefGoogle Scholar
Ficht S, Dose C, Seitz O (2005) As fast and selective as enzymatic ligations: unpaired nucleobases increase the selectivity of DNA-controlled native chemical PNA ligation. Chembiochem 6:2098–2103PubMedCrossRefGoogle Scholar
Dose C., Ficht S., Seitz O (2006) Reducing Product Inhibition in DNA-Template-Controlled Ligation Reactions. Angew Chem 118, 5495-5499; Angew Chem Int Ed 45, 5369–5373CrossRefGoogle Scholar
Ficht S, Mattes A, Seitz O (2004) Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates. J Am Chem Soc 126:9970–9981PubMedCrossRefGoogle Scholar
Grossmann TN, Strohbach A, Seitz O (2008) Achieving turnover in DNA-templated reactions. Chembiochem 9:2185–2192PubMedCrossRefGoogle Scholar